PROJECT SUMMARY. This research seeks to understand spatial phosphoinositide signaling (PI) mechanisms in the cytosol and nucleus. These pathways have broad implications for cancer, neurodevelopmental disorders, diabetes, and several congenital diseases. In the cytosol, agonists, such as EGF (and many others), activate signaling pathways that control most cellular functions. In the nucleus, these pathways are separate from known membrane compartments but control stress responses that impact DNA repair, cell survival, and other events. Agonists activated PI3K signaling occurs through the IQGAP1 scaffold that assembles multiple pathways including the PI 3-kinase and Erk pathways. Yet, how the IQGAPs assemble specific signaling pathways is not understood. We will focus on the assembly of the full PI 3-kinase pathway on IQGAPs. This includes the assembly of the PI 4-kinase (PI4KIII?), type I? PIP 5-kinase (PIPKI?), PI3K, Ras, PDK1 and Akt into the scaffold. Remarkably, we show that IQGAP2 and IQGAP3 also assemble the PI 3-kinase pathway components but with different outcomes. IQGAP2 is tumor suppressor in cancer cells whereas IQGAP1 and IQGAP3 promote PI3K signaling and cell proliferation. Here, we will explore how receptors stimulate the assembly of the IQGAPs signaling pathways with an emphasis on the EGF receptor and IQGAP1-PI 3-kinase and Erk pathways. We will emphasize spatial PI 3-kinase signaling at endosomal compartments at proximity to microtubules by linkage with microtubule associated protein 4 (MAP4) that interacts with IQGAP1 and PI 3-kinase. The link between the cytosolic and nuclear PI signaling is the PIPKI?, which generates PIP2 in the cytosol and nucleus Nuclear PI signaling remarkably is not associated with membrane compartments. We showed that a nuclear poly(A) polymerase, Star-PAP (for speckle targeted PIPKI? regulated-poly(A) polymerase), associates with PIPKI? and is activated by phosphatidylinositol-4,5-bisphosphate (PIP2). Star-PAP controls ~40% of genes and is regulated by many signals. Recently, we have shown that PIPKI? also binds to the tumor suppressor p53, and that p53 is a PIP2 effector. The binding of PIP2 stimulates p53?s interactions with other nuclear factors that control p53 function. Both Star-PAP and p53 are also regulated by inositol polyphosphate multikinase (IPMK) that generates phosphatidylinositol-3,4,5-trisphosphate (PIP3) associated with p53, and nuclear PTEN that dephosphorylates this PIP3. We have identified Star-PAP and p53 as two key effectors of nuclear phosphoinositide signaling during stress signaling. This proposal will focus on the mechanism and impact of this stress pathway on Star-PAP functions. Remarkably the PIPn is so tightly associated with Star-PAP and p53 that it is stable to SDS-PAGE suggesting a covalent linkage and we will explore how PIP2 is linked to Star-PAP and p53. Is this covalent or a very tight interaction that is resistant to denaturation? Our findings indicate new avenues for potential therapeutic control of both the cytosolic and nuclear PI pathways as these pathways have fundamental implications in many disease processes but with an emphasis on cancer.